To study the dynamics of individual molecules over an extended period of time, molecules must first be localized in space. We accomplish this by immobilizing them on the surface of a microfluidic cell. The procedure starts with the assembly of the cell, which contains four chambers where the biomolecules will be fixed.
The chambers are connected to the exterior through inlet and outlet capillaries. The cell is made by fusing a patterned PDMS disc to a quartz cover slip. The assembled cell is then mounted on the fluorescence microscope and flexible tubings are connected to the capillaries to allow the easy exchange of solutions through the chambers to activate the surface for molecule immobilization first flow, a solution of biotinylated BSA through one of the channels.
BSA will absorb to the chamber. Surfaces then flow a solution of streptavidin, which will in turn bind to the biotin. Finally, flow your biotinylated oligonucleotide, which will bind to the streptavidin, thus anchoring the molecule of interest to the surface of the quartz cover slip.
An imaging buffer designed to maximize the lifetime stability and intensity of the fluoro fours is flowed through the chamber at a constant rate throughout the entire acquisition period. The automated setup scans different sections of the surface for molecules having both donor and acceptor flora fours, and then moves them one by one into the probing volume obtaining for each a fluorescence intensity time trace until both Fluor fours photo bleach. Hi, I'm Nicholas Dfi from the laboratory of Professor Miller in the biomedical engineering department at Boston University.
Today we will show you a procedure to obtain single molecule F traces from immobilized nucleic acids using a confocal setup. We use this procedure in our laboratory to study nucleic acid dynamics and nucleic acid and enzyme interactions. So let's get started.
The Microfluid Excel is made by fusing a patterned two millimeter thick poly dimethyl silicone or PDMS disc to a freshly cleaned quartz cover slip. To make the patterned PDMS disc combined 10 parts of silicone elastomer base with one part curing agent mix well and Degas for one hour in a vacuum. After the elastomer base has been Degas, pour it gently into the microfluidic cell mold.
Leave the elastomer mix to cure on a hot plate at 70 degrees Celsius for two hours. When cured, carefully peel the PDMS disc off from the mold using a razor blade. The PDMS disc contains four 30 microliter rectangular chambers.
Fuse the flat side of the PDMS disc, the side not containing the channels, onto a one inch glass window by plasma oxidizing both surfaces for 30 seconds. The glass window contains eight to two millimeter holes at both ends of the channels which were previously drilled. Next, a two inch long flexible fused silica capillary tubing is inserted through each glass window hole.
To do this first insert a needle and then insert the capillary tubing until it reaches the channel. Seal the glass window holes using a fast curing silicone casting compound such as quick cast. Rinse the channels with ethanol and water after plasma activating the cell and a freshly piranha cleaned one inch circular quartz cover slip for 30 seconds.
Fuse the cover slip to the side of the cell containing the channels. The chambers are now sealed. Leave the assembled cell curing overnight at room temperature.
After the assembled microfluid Excel has cured overnight, it is placed in a custom made holder and mounted on the stage of the confocal microscope to begin activating the surface of the cell. For molecule immobilization, connect flexible tubing to the capillaries of the cell to facilitate buffer injection using a syringe and a needle. Now inject a solution of 0.1 milligrams per milliliter biotinylated BSA in buffer A into the channel and incubate for at least 30 minutes.
After the 30 minute incubation flow. 300 to 500 microliters of buffer A To remove any unabsorbed biotinylated, BSA from the channel, be careful not to trap any air bubbles in the chamber. Next, inject a solution of 0.1 milligrams per milliliter STR din in buffer A and incubate for 15 minutes.
After the 15 minute incubation flow, 300 to 500 microliters of buffer A to remove any unabsorbed strep din from the channel. As before, avoid trapping any air bubbles in the chamber. Finally, flow a solution of three to five picomolar of the biotinylated sample.
To check the coverage of fluorescent molecules, a surface scan is performed. Incubate the biotinylated sample for 10 minutes and repeat the flow of buffer. A As shown previously, the imaging buffer or IB consists of an enzymatic oxygen scavenging system and a triplet quencher such as trol locks to make the IB first dissolve.
Five milligrams of trol locks into 10 milliliters of glucose buffer by vortexing for a couple of minutes and shaking for at least 10 minutes. Filter the solution and leave it to gassing in a vacuum for 10 minutes. Gently mix the TRO lock solution with the Glocky enzymatic solution while avoiding the formation of air bubbles.
Fill the reservoir with the IB solution. Then using a mechanical syringe pump control the flow rate, flow the IB through the channel at a constant rate of five microliters per minute, bubble argon gas into the IB reservoir to prevent atmospheric oxygen from dissolving into the buffer. The surface scanning, molecule localization and acquisition of single molecule intensity traces is controlled by the lab view program, which scans 20 by 20 micrometer squared areas at 0.2 micrometer resolution with a rate of 0.1 micrometer per millisecond.
The data from the surface scanning is immediately processed to yield the intensity weighted locations of all pixels above the background counts. Once the immobilized molecules are found, they are moved one by one into the confocal volume and the intensities of the donor and acceptor fluorophore are recorded as a function of time until both flora fours photo bleach. The stage is then programmed to move to a new origin, 100 millimeters away and the scanning process is repeated.
The assembled MICROFLUID Excel is shown here. These are surface scans showing single molecules in the green donor and red acceptor channels. Some typical single molecule traces showing the acceptor intensity in red and donor in green are shown.
We've just shown you how to obtain single molecule traces from immobilized biomolecules using a confocal setup. When doing this procedure, it's important to remember to do every step carefully and take your time since this is a challenging experiment. So that's it.
Thanks for watching and good luck with your experiments.
